Throughout this application, various publications are referenced in parentheses. Full citations for these publications can be found immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
It has long been well known that the salts of butyric acid, particularly the sodium salt, act in vitro on many abnormal or transformed cell lines to cause these cells to change to a more normal state, including phenotype and function. This "differentiation" action is also shown by a number of agents with no readily obvious structure-activity relationship. In vitro, butyrate acts on some cancer or leukemia cancer cell lines such as HT-29, or HL-60 in this fashion, when maintained at concentrations of about 0.3 to 5 millimolar (mM). Desirable changes of the aberrant blood cells of sickle cell anemia and thalassemia can be obtained in vitro at lower concentrations of about 0.05 mM [Perrine S. P., et al., N. Engl. J. Med., Vol. 328, pages 81-86, (1993); Perfine, S. P., et al., Blood, Vol. 74, pages 454-459 (1989)].
Attempts to utilize butyrate salts in therapy have not generally been successful. An early attempt at using intravenous infusions of sodium butyrate at 500 mg/kg body weight per day for several days produced only a short-lived remission in a child with leukemia [Novogrodsky, A., et al., Cancer, Vol. 51, pages 9-14]. A larger study [Miller, A. A., et al., Eur. J. Cancer Clin. Oncol., Vol. 23, pages 1283-1287 (1987)], using the same infusion rate showed no clinical response, but also demonstrated that the infused butyrate had a very short metabolic half-life of about 6 minutes, resulting in peak blood levels below 0.05 mM, considered ineffective for leukemia. Higher rates of intravenous infusions could not be considered because of the risk of toxicity from sodium overload, and achieved success in treating thalassemia and sickle cell patients by continuous intravenous infusions of 500 mg/kg/day or higher doses, if needed, for several days. In these studies also, blood levels of butyrate did not exceed 0.05 mM and the butyrate was apparently rapidly metabolized [Perrine, et al. (1993)].
Butyrate is a normal metabolite supplied to mammals from 2 main sources. It is produced as a major product of bacterial fermentation of unabsorbed carbohydrate in the colon, and reaches concentrations of up to 20 mM in the colon and feces of animals and man [Cummings, J. H., Gut, Vol. 22, pages 763-779 (1981)]. The other source of butyric acid is the diet, where it is present at low levels in many fruits and vegetables, but its richest source is from milk fat (butter, etc.) which contains 3-4% butyrate in a complex of glycerides, or esters of glyceryl [Composition of Foods: Dairy and Egg Products (1976), U.S. Dept. Agriculture Handbook 8-1, Washington, D.C.]. However, when butyryl triglyceride (tributyrin, or glyceryl tributyrate) was used at 5% of the feed in our laboratories as means of preventing chemically induced colon cancer in mice, it was ineffective [Deschner, et al., Cancer Letters, Vol. 52, pages 79-82 (1990)] although it did not produce the promoting or cancer increasing effect of the sodium salt used by others in equivalent intake [Freeman, H. J., Gastroenterology, Vol. 91, pages 596-602 (1986)]. Apparently, the high sodium intake acted as a cancer promoter [Freeman, (1986)], and the butyrate was metabolized too rapidly to be preventive.
The object of this invention, therefore, is a method of administering butyric ester compounds to a subject suffering from a disease against which butyrate would be effective, which would provide for an effective amount of butyrate to remain in the subject's system for an effective period of time and thereby treat the disease.
It was hypothesized that butyryl glycerides, including but not limited to tributyrin, several possible dibutyl glycerides and monobutyrin such as the 1-butyryl glyceryl, which are essentially non-charged, i.e., non-anionic, substances of comparatively low molecular weight, administered in an oral bolus dose, are potentially easily absorbable directly into the stomach and upper gastrointestinal tract. They can then enter into the lipid transport system, and be slowly hydrolyzed by the lipases in serum and possibly liver. These lipases are generally slower than the gastric and pancreatic lipases of the G.I. tract. The net effect of oral bolus administration would be to maximize systemic absorption, such as from the stomach, of one or more of the glycerides, as well as butyric acid, which would then act as a "reservoir" of butyrates for slow release within the blood stream. The unexpected activity of the glycerides per se as differentiating agents could add significant therapeutic effect to the total.